In Alzheimer's disease (AD), the microtubule-associated protein tau is hyperphosphorylated and aggregates into paired helical filaments (PHFs), leading to NFT formation. Previously, we described a cascade of molecular events associated with NFT formation within cortical neurons in AD. Whether similar or different events occur during NFT formation within cholinergic basal forebrain (CBF) projection neurons during the progression of AD remains unknown. The Program Project entitled Neurobiology of Mild Cognitive Impairment in the Elderly provides an outstanding opportunity to extend these findings to an early stage of AD, people who have died with a clinical diagnosis of mild cognitive impairment (MCI). Establishment of this cascade of tau changes is paramount as NFTs provide a window into the tau portion of the neruonal degnerationn in AD and will allow for in vitro modeling to approach a mechanistic understanding of tau pathology and and aggregation. We propose to investigate whether formation of fibrillar pathologies follows a definable sequence of molecular events that directly impact tau's assembly competency through phosphorylation and truncation while other events (tau cleavage by Puromycin-Sensitive Aminopeptidase expression and non-canonical tau isoform expression) inhibit this process in CBF neurons during the early stages of AD. We propose to place tau changes in an overall molecular context within affected CBF neurons by correlating NFT evolutionary states with enzyme and other proteins expression profiles affecting tau association and function. Specifically: 1. We will test the hypothesis that CBF neuron NFT formation occurs in a linear and orderly fashion assayed and ordered by alterations in phosphorylation and truncation events as certain phosphorylation and carboxy-terminal truncation events appear facilitative of NFT formation; 2. We will test for the expression of candidate enzyme genes known to stimulate or inhibit tau's ability to form NFTs defined by specific antibodies to phosphoepitopes, conformational states, and C-terminal truncation sites and, 3. We will test the hypothesis that the expression of small tau isoforms lacking the microtubule binding repeats and carboxy termini inhibit tau filament (and hence NFT) formation. These aims will begin to define the molecular milieu of tau tangle formation and provide insight into regulatory mechanisms involved in controling and/or inhibiting this phenomenon, which are needed to develop new treatments for AD.